The present invention relates to a process for the isolation of compound scopoletin useful as a nitric oxide synthesis inhibitor from the plant Artemisia annua and other plant families. Scopoletin belongs to the coumarin group of compounds. Scopoletin (7-hydroxy-6-methoxy coumarin is a derivative of benzo-xcex1-pyrone coumarin and is found in plants both in the free state and glycosides.
Natural coumarins exert varied and pronounced effects on living organisms. Coumarins exhibit numerous pharmalogical and physiological activities such as antibacterial, vasoditory and diuretic effects, anticoagulant properties, hepatoprotective and respiratory stimulation. Scopoletin showed very good growth inhibitory properties. (Shilling D G and Yoshikawa F, Amerchem. Soc. Syinp Series, 330:335-342). Recently, it was reported that scopoletin showed inhibition of nitric oxide (NO) synthesis in murine macrophages. Macrophages play a major role in last defense against infection and tumor development and this activity is regulated through the production of several mediates. The production of NO by macrophages mediates killing or growth inhibitors of tumor cells, bacteria fungi and parasites. Therefore it will be valuable to develop a potent and economic source of scopoletin, inhibitor of NO for potential therapeutic and commercial use in the future (Tai-Hyun Kang et al: scopoletin: an inducible NO synthesis inhibitory active constituents from Artemisia feddi, Planta Medica, 65:400-403, 1999); Invitro inducible NO synthesis inhibitory active constituent from Ttraxinus rhynchobhyeli, Plant Medica, 65:656-658, 1999).
Scopoletin is widely distributed in the plant kingdom and is isolated from the different parts (roots, fruits, leaves, stems, etc.) of the plant, but several related members frequently are found together making their isolation difficult. In general isolation of coumarins depends initially upon successive extraction of dried plant with commonly used solvents of increasing polarities (pet ether, benzene, ether, acetone or methanol).
The conventional method used for the isolation of the coumarins used dried powder material extracted with 95% methanol/ethanol, and the concentrated extract was treated with 10% alkali solution. The alkaline solution was extracted with ether to remove the fatty material. The aqueous solution was acidified and extracted with ether or chloroform to obtain crude coumarins. Further, the purification was carried out on column chromatography over silica gel or alumina to get pure coumarins. Certain coumarins (e.g. umbilliferon, scopoletin) can be isolated from plant tissues by sublimation methods (Abu-Mustafa, E. A.; El-Tawil, B. A. H. and M. B. E. Fayez; Phytochemistry, 3, 701 (1964)).
However, the method of isolation of coumarin through acid based treatment is not ideal because it is hardly possible to avoid some degradation of the coumarins which lead to the isolation of artefacts rather than the original compound or the loss of the compounds. In another process purification done by sublimation may involve thermal degradation with formation of artefacts. The overall yield of recovery is reduced. As such no plant with high content of scopoletin for commercial exploitation nor any large scale process for the isolation of scopoletin has been reported.
The objective of the present invention is to explore new plant/plant part with a high content of scopoletin.
Another objective of the invention is the selection of solvent for the selective extraction of scopoletin.
Still another objective of the invention is to develop an economic process for the isolation of pure scopoletin.
Accordingly, the present invention provides a process for the isolation of nitric oxide synthesis inhibitor compound scopoletin from the plant Artemisia annua, and other plant families, which comprises the extracting the powder plant parts with an aqueous acetonitrile solvent, partitioning with a chlorinated solvent, evaporating the solvent, crystallizing a residue from the solvent and employing chromatography to yield pure scopoletin.
Novelty of the Process
1. Selection of the plant part for the extraction of scopoletin improved the yield and reduced the processing cost.
2. The extraction of the plant part with aqueous acetonitrile is more economic with a better yield of scopoletin as compared to the use of other polar solvents like methanol or ethanol.
3. In this process, by avoiding any acid base treatment for the isolation of scopoletin the yield was improved.
4. The selective transfer of scopoletin from the aqueous extract to the halogentaed non polar phase resulted in easy purfication and isolation of pure scopoletin.
5. The purificaton of the crude extract resulting in crystallisation of 50% of scopoletin before the chromatography.
6. The process allows the reuse of solvents and silica gel.
7. These advantages are of significant economic values for large scale production of scopoletin.
The present invention provides a process for the isolation of compound scopoletin which is used as nitric oxide synthesis inhibitor from Artemisia annua and other plant families, said process comprising:
a) extraction of dried powdered material of different plant parts with an aqueous acetonitrile solvent in the ratio of 1:5 for 6 to 8 hrs.,
b) concentration of the extracted solvent upto 30% of its original extract under vacuum,
c) partitioning the concentrated extract with halogenated solvent to transfer scopoletin in the non-polar halogenated solvent,
d) drying halogenated solvent over anhydrous sodium sulphate and evaporating the solvent,
e) crystallizing the residues in methanol and filtering the crystals,
f) concentrating the filtrate and chromatographed on silica gel,
g) eluting scopoletin in chloroform methanol mixture; and crystallization of the fractions containing the scopoletin to get the pure scopoletin compound.
In one embodiment of the invention, Artemisia annua plant was selected for the isolation of scopoletin. Artemisia annua ariel parts from Spain have reported 0.02% scopoletin and England (cult) 0.034%. China has also reported the presence of scopoletin from the whole plant (Brown, G. D.: Two new compounds from Artemisia annua: J.Nat. Prod. 58, 300 (1992); Marco, J. A.; Sawz, J. F.; Bea, J. F.; Barber, O.: Phenolic constituents from Artemisia annua: Pharmazie 45, 382-383 (1990); Liu, H. M.; Li, G. L., Wu, H. Z.: Studies on the constituents of Quinghao:Yao Hsuch Hsuch Pao, 37, 129-143 (1979)).
In India CIMAP, Lucknow, has developed a new variety of Artemisia annua xe2x80x9cJeevan rakshaxe2x80x9d producing high content of artemisinin and biomass (stems and leaves). Artemisinin and its derivative are reported as potent against chloroquine resistant multi drug resistant and severe complicated malaria. Artemisia annua plant is the only source of artemisinin.
Screening of all the three major parts of the A. annua plant of the new variety for scopoletin was carried out by HPLC. The content of scopoletin obtained in different plant parts are as follows; leaves 0.2%, Stems 0.3% and roots 0.004%. The yield of the scopoletin is very high as compared to other reported plants. The stem part of the plant Artemisia annua is a waste material as no artemisinin is present in the stems. The biomass of the stem portion of the plant is five times more than of the leaves. Therefore, we have selected the stem part of the plant Artemisia annua for the isolation of scopoletin. Also the stem portion of the stems was found to contain less colouring and fatty material which eases the isolation and purification of scopoletin.
In the prior art, it has been observed that non polar solvents for extraction of the plant materials were employed, resulting in less recovery of coumarins. Still, polar solvents (methanol and ethanol) used for the extraction of coumarins resulted in a higher amount of total extract having more colouring and fatty material. The separation of fatty and colouring matter is a difficult task. In the present invention, we have selected aqueous acetonitrile solvent for the extraction which yielded a higher amount of scopoletin with less amount of colouring and fatty material. Also, the separation of water from acetonitrile for recovery of the pure solvent for reuse is much easier.
In the prior art the scopoletin was purified from the crude extract through acid base treatment or by sublimation method which reduces the amount of scopoletin due to rearrangements and thermal decomposition. In the present invention of the improved process, selective transfer of the coumarins from the aqueous extract into the non polar phase was carried out by partitioning the aqueous phase with chlorinated solvent (Carbon tetra chloride, di chloromethane, chloroform). By employing this step, most of the colouring and fatty material is left in the polar phase thereby enriching the scopoletin in the non polar phase which is easily crystallisable (50-60%) in the crude extract itself The scopoletin left in the mother liquor after crystallization is subjected to column chromatography over silica gel in ratio of only 1:10 for complete isolation of the pure scopoletin. The partition of scopoletin from aqueous extract to non polar solvent reduces the bulkiness of the crude extract by 60-70% which in terms requires less amount of silica gel and solvent in the process.
The process consists of the following operations
1. Shade drying and grinding of the stems of Artemisia annua. 
2. Extracting the powdered Artemisia annua stems with aqueous acetonitrile solvent by cold percolation.
3. Concentrating the total extract under vacuum.
4. Partitioning of the aqueous acetonitrile phase with halogenated solvent.
5. Removal of moisture from the total halogenated extract.
6. Distillation of the halogenated solvent for obtaining the residual extract.
7. Crystallisation of the scopoletin from the residual extract.
8. Filtration and concentration of mother liquor.
9. Column Chromatography of mother liquor over silica gel for recovery of pure scopoletin.
The present invention is to provide a process for the extraction and isolation of scopoletin from the plant Artemisia annua to overcome the drawbacks of the hitherto known process. The invention more particularly provides a process which gives a cheaper and higher yield of nitric oxide synthesis inhibitor compound scopoletin from the natural source.
Accordingly, the present invention provides a process for the extraction and isolation of scopoletin from Artemisia annua which comprises extraction of a plant part, preferably dried stem powder of Artemisia annua with aqueous acetonitrile solvent in the ratio of 1:5, concentration of the extract under a vacuum, partitioning of the concentrated extract with a non polar halogenated solvent, distillation of the halogenated solvent, crystallization of the residue in methanol, filtration of scopoletin, concentration of the mother liquor and performing chromatography over silica gel for obtaining pure scopoletin.
In an embodiment, of the invention the solvent used for the extraction is selected in different ratios of acetonitrile:water from 1:9 to 9:1.
In another embodiment, of the invention the halogenated solvent used for partitioning is to be selected from dichloromethane, carbon tetra chloride, chloroform etc. In another embodiment of the invention the plant part for extraction of scopoletin is selected from stems, leaves, roots etc.
In another embodiment, of the invention the separation of scopoletin over silica gel whereas the ratio of crude extract to silica gel is selected from 1:5 to 1:20 preferably either from 1:5, 1:10 or 1:20.
The details of the invention provided in the following examples are given by way of illustration only should not be construed to limit the scope of the present invention
Yet another embodiment of the invention, extraction and isolation of scopoletin is from plant families such as Umbelliferae, Rutaceae, Compositae, Leguminosae, Moraceae, Caryophyllacae, etc.
Still another embodiment of the invention, the scopoletin is crystallized in the solvent which is selected from chloroform, acetone, methanol and mixtures thereof.
One another embodiment of the invention, scopoletin isolated from different parts of Artemisia annua is in the range of 0.25-0.30% in stem, 0.16-0.20% in leaves, and 0.003-0.004% in roots.